Single chain antibodies against beta-amyloid peptide

ABSTRACT

Anti-AβP scFvs and single domain antibodies were generated, and when these antibodies were displayed on filamentous phage or as soluble protein molecules, stabilized by the maltose binding protein, they could prevent the fibrilization of Aβp 1-40 and disaggregate Aβp 1-40 fibrils generated in vitro. The anti-Aβp scFv antibodies also stained amyloid neuritic plaques on slices from transgenic mice. The anti-AβP scFv and single domain antibodies can be used for inhibiting or treating Alzheimer&#39;s disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of application Ser. No. 12/064,144, filed Oct. 29, 2008, which claims the benefit of priority from provisional U.S. application No. 60/60/709,102, filed Aug. 18, 2005, the entire content of which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antibodies for treating Alzheimer's patients.

2. Description of the Related Art

Beta amyloid peptide (Aβp), the product of processed amyloid precursor protein, is accumulated in the brains of Alzheimer patients for reasons that have not been elucidated yet. Immunotherapy towards Aβp is one approach researchers are using in looking for a relief or a cure for this disease. Antibodies generated towards the N-terminus of the beta amyloid peptide prevented the fibrilization of Aβp peptide in vitro, and significantly reduced the cytotoxic effects of Aβp fibrils on PC12 cells (Frenkel et al., 2000). Also, immunization with Aβ of transgenic mice that express the human amyloid precursor protein (APP) and exhibit Alzheimer's-like pathology, demonstrated that the mice developed humoral reaction towards the antigen, and exhibited a significant reduction in plaque load compared to controls (Schenk et al., 1999). Moreover, in these mice, active clearance of amyloid by microglial cells was noted. Bacskai et al. (2002) reported that FITC-labeled F(ab)₂ fragments of monoclonal anti-Aβ antibody or full-length antibody led to the clearance of 45% of the amyloid deposits in 18-month-old transgenic mice within 3 days. These results suggest that immunotherapy has the potential to either delay the generation of or reduce one of the major hallmarks of Alzheimer's pathology, i.e., Aβ plaques.

Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.

SUMMARY OF THE INVENTION

The present invention provides single chain antibodies against β-amyloid peptide, which include the complementarity-determining regions of the heavy antibody chain and/or light antibody chain, preferably single chain (scFv) or single domain antibodies, and nucleic acid molecules and vectors encoding these anti-Aβp antibodies. Also provided are a host cell transformed with the nucleic acid molecule encoding the antibody against β-amyloid peptide and a method for producing and isolating such an antibody.

The present invention further provides a composition and a method for inhibiting or treating Alzheimer's disease by administering an anti-Aβp antibody of the present invention to a patient in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the sequences of the linker forward (SEQ ID NO:1) and reverse (SEQ ID NO:2) primers, the linker sequence (SEQ ID NO:3) and the linker peptide sequence (SEQ ID NO:4).

FIG. 2 is a schematic diagram of an outline of the procedure for heavy (H) and light (L) chain synthesis and their connection with the linker sequence.

FIGS. 3A-3D show the nucleotide and amino acid sequences of RCK37 (SEQ ID NOs:5 and 6; FIG. 3A) and RCK22 (SEQ ID NOs:7 and 8; FIG. 3C) along with their CDR nucleotide and amino acid sequences (SEQ ID NOs:9-32; FIGS. 3B and 3D).

FIG. 4 is a graph of the results of an ELISA assay demonstrating the binding of Q-RCK37, Q-RCK22 and Q-helper to three different antigens which all harbor the EFRH (SEQ ID NO:33) epitope. The results are depicted as OD measurements at 492 nm.

FIGS. 5A-5C show amyloid plaque stained brain sections of hAPP tg mice where FIG. 5A is staining with Q-RCK22, FIG. 5B is staining with Q-RCK37, and FIG. 5C is staining with Q-helper (no ScFv).

FIG. 6 is a graph of the results of an ELISA assay demonstrating the binding of soluble MBP-ScFv to β-amyloid peptide and the binding of MBP alone as a control.

FIGS. 7A-7D are graphs showing the results of the ELISA assay performed with constant β-amyloid concentrations and elevated 37H or 37L concentrations. The binding of different concentrations of 37H (FIG. 7A) and 37L (FIG. 7B) to 10 μM Aβp 1-40 and the binding of the 12.5 μM 37H (FIG. 7C) and 37L (FIG. 7D) to different concentrations of Aβp 1-40 are shown.

FIGS. 8A and 8B show the binding of 37H to β-amyloid fibrils in solution and their partial disaggregation. In FIG. 8A, the Tht emission values at 485 nm as a function of antibody added to the pre-aggregated β-amyloid are shown. The same results are shown in FIG. 8B except that the results are depicted relative to the level of Aβp 1-40 Tht emission which is considered 100% aggregated.

FIG. 9 shows Tapir staining of amyloid plaques in a PDAPP mouse brain section by 37H nanobody.

FIG. 10 is a graph showing amyloid load as determined by Thioflavin S staining. Data are presented as the average scores of all mice in each group: n=12 in the scFv treated groups and n=11 in the control group.

FIG. 11 is a graph showing amyloid load determined by staining with 21F12, an anti-Ab 1-42 antibody. Data are presented as the average scores of all mice in each group: n=12 in the scFv treated groups and n=11 in the control group.

DETAILED DESCRIPTION OF THE INVENTION

Primers that can be used to amplify mouse antibody variable heavy and light chains as well as primers to connect these chains by a peptide linker and primers to amplify the assembled antibodies were generated. These primers were used to generate a scFv library from the spleens of immunized mice and from hybridoma 196 that expressed anti-AβP antibody. Novel scFv antibodies were isolated from these sources and designated as RCK37 and RCK22, respectively. These antibodies when displayed on filamentous phage or as a soluble protein molecule, stabilized by the maltose binding protein, can prevent the fibrilization of Aβp 1-40 and can disaggregate Aβp 1-40 fibrils generated in vitro. They also stained amyloid neuritic plaques on slices of transgenic mice. Single domain antibodies with only the heavy or light chain of RCK37 were generated and were also shown to prevent the fibrilization of Aβp 1-40 and can disaggregate Aβp fibrils generated in vivo.

The present invention provides an antibody against the β-amyloid peptide. The anti-Aβp antibody according to the present invention contains the complementarity-determining regions (CDRs) of an antibody heavy chain or light chain, where the CDRs are the hypervariable regions of an antibody or immunoglobulin molecule that recognize and bind an epitope. The CDRs contained by the anti-Aβp antibody according to the present invention are a combination of CDR1, CDR2, and CDR3 sequences selected from: (1) SEQ ID NOs:10, 12 and 14 (heavy chain CDRs of RCK37); (2) SEQ ID NOs: 16, 18 and 20 (light chain CDRs of RCK37); (3) SEQ ID NOs: 22, 24 and 26 (heavy chain CDRs of RCK22); and (4) SEQ ID NOs: 28, 30 and 32 (light chain CDRs of RCK22).

Preferably, the anti-Aβp antibody of the present invention is a single chain (scFv) antibody or a single domain (heavy or light chain of Fv) antibody. When the antibody is a single domain antibody, it has the combination of CDR sequences of (1), (2), (3) or (4) above and when the antibody is a scFv antibody, it has the combination of CDR sequences of (1) plus (2) or (3) plus (4). Most preferably, the single chain scFv antibody according to the present invention has the amino acid sequence of either SEQ ID NO:6 (RCK37) or SEQ ID NO:8 (RCK22) and the single domain antibody according to the present invention has the amino acid sequence corresponding to residues 1 to 124 or residues 140 to 247 of SEQ ID NO:6.

The present invention is also directed to a nucleic acid molecule which encodes the anti-Aβp antibody of the present invention. Preferably, the nucleic acid molecule includes the nucleotide sequence of either SEQ ID NO:5 or SEQ ID NO:7 when the nucleic acid molecule encodes a single chain scFv antibody or includes nucleotides 1 to 372 or 418 to 741 of SEQ ID NO:5 when the nucleic acid molecule encodes a single domain antibody.

The present invention further provides a vector which contains the nucleic acid molecule encoding the anti-Aβp antibody of the present invention, preferably as an expression vector capable of expressing the anti-Aβp antibody in a host cell.

Further aspects of the present invention include a host cell transformed with the vector of the present invention and a method for producing and isolating the anti-Aβp antibody of the present invention. This method of production involves culturing the transformed host cell to express and produce an anti-Aβp antibody and then isolating the produced anti-Aβp antibody from the cell culture.

A composition, preferably a pharmaceutical composition which contains an effective amount of the anti-Aβp antibody of the present invention and a pharmaceutically acceptable carrier, diluent, excipient or auxiliary agent, is also provided by the present invention.

Finally, the present invention also further provides a method for inhibiting or treating Alzheimer's disease by administering, preferably intranasally, the anti-Aβp antibody of the present invention by passive immunization to a patient in need thereof to inhibit or treat Alzheimer's disease. Antibodies and methods for passive immunization against Alzheimer's disease and other diseases or disorders characterized by amyloid aggregation are well known in the art. See for example WO 99/27944 and U.S. Pat. No. 5,688,651, the entire contents of each of which are incorporated herein by reference.

Having now generally described the invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration and is not intended to be limiting of the present invention.

Example 1 ScFv Antibodies Against β-Amyloid Peptide

In the study in this example, mice were immunized with MAP-(EFRH)₂ and an ScFv library displayed on filamentous phage. MAP is an abbreviation for the multiple antigen peptide presentation disclosed in WO 2003/076455. Anti-EFRH antibodies were selected by biopanning. Also, a single chain antibody was generated from Hybridoma 196 that express antibodies against EFRH (SEQ ID NO:33). The best binding ScFv antibodies were either displayed on filamentous phage or produced as soluble MBP (maltose binding site)—fused antibodies and used for further investigation.

Materials and Methods

mRNA Extraction

Mice were immunized with MAP-EFRH₂ and developed high titer of antibodies to the EFRH (SEQ ID NO:3) epitope. The spleens of 5 mice were excised, cut into small pieces and homogenized in RNA extraction reagent (tri-reagent, Biological Industries, Kibbutz Mishmar Haemek, Israel). RNA was extracted according to the manufacturer's instructions, precipitated and suspended in dH₂O. mRNA was extracted using μMacs mRNA isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

RNA and mRNA were also extracted from hybridoma cells that expressed monoclonal antibodies against the EFRH (SEQ ID NO:33) epitope using the same procedures as above.

Primer Design

Mouse variable heavy and light chains sequence-alignments were used from the interface of the Kabat data bank (kabatdatabase.com) and the Antibody Group (ibt.unam.mx/vir). Degenerated primers were designed according to consensus amino acids in the amino- and carboxy-termini of the variable heavy and light chains (Table 1). The sequences of the forward and reverse primers of the heavy and light chains were used to design the other primers. Linker primers to join the heavy and light chains were designed with an NheI site. The linker-rev primer contains at its 5′ end about half of the peptide-linker-encoding sequence while the second half-linker sequence is contained in primer linker-fwd. The two primers overlap at 24 nucleotides out of the 48 nucleotides that encode the linker peptide (FIG. 1). Primer NcoI-fwd contains at its 5′ an NcoI restriction site and degenerated sequence of the 5′ end of the heavy chain. Primer NotI-rev contains the sequence for NotI nuclease and the degenerated sequence of the 3′ end of the light chain.

TABLE 1 Library primers VH-fwd (23 mer) 5′-SANRTBMARYTKSWGSAGYCWGG-3′ (SEQ ID NO: 34) VH-rev 24 mer 5′-TGMRGAGACNGTGASHRDVGTBCC-3′ (SEQ ID NO: 35) VK-fwd 23 mer 5′-GANRTYKTGMTSACVCARDCTMC-3′ (SEQ ID NO: 36) VK-rev 24 mer 5′-MCGWTTBAKYTCCARSTTKGTSCC-3′ (SEQ ID NO: 37) Linker primer-fwd (47 mer) 5′-TCAGGGGGAGGT

GGTGGCGGAGGCTCTGAIRTYKTGMTS ACICA-3′ (SEQ ID NO: 1) Linker primer-rev (47 mer) 5′-GCCACC

ACCTCCCCCTGATCCGCCTCCACCTGMRGAGAC IGTGASIRIIGT-3′ (SEQ ID NO: 2) NcoI primer (fwd) (30 mer) 5′-CATGCCATGGCTSANRTBMARYTKSWGSAG-3′ (SEQ ID NO: 38) NotI primer (rev) (33 mer) 5′-ATAGAATGCGGCCGCMCGWTTBAKYTCCARSTT-3′ (SEQ ID NO: 39) Glossary B = C or G or T D = A or G or T H = A or C or T K = G or T M = A or C N = A or C or G or T R = A or G S = C or G V = A or C or G W = A or T Y = C or T I = inosine

Library Construction

All the procedures described below have been carried out separately with spleen mRNA and with hybridoma mRNA. Synthesis of the first strand cDNAs of the heavy and light chains variable domains were carried out with primers linker-rev and Not-rev, respectively, using RevertAid synthesis kit (Fermentas, Vilnius, Lithuania) (see procedure outline in FIG. 2). Primer NcoI-fwd was added to the V_(H) reaction and primer Linker-fwd was added to the V_(K) reaction. Amplification of the heavy and light chains, each with an extended linker-half was carried out using AmpliTaq DNA polymerase (Perkin Elmer). The heavy and light chains were gel purified and digested with NheI followed by digestion of V_(H) fragments with NcoI and V_(K) fragments with NotI (these two sites are contained in the 5′ region of primers NcoI-fwd and NotI-rev, respectively). For the spleen library, 0.8 μg of each of the digested chains were combined with 1 μg pCANTAB5E (Amersham BioSciences, Piscataway, N.J.) vector that was modified to contain an NcoI site and was linearized by NcoI and NotI digestion. For the hybridoma library, 50 ng of each fragment and vector were ligated. Tri-partite ligations were performed using T4 DNA ligase for 2 hr at room temperature. The ligated mixtures were transferred into E. coli TG1 cells by electroporation. Transformants were selected on 2YT agar plates with 100 μg/ml carbenicillin for 12 hr at 30° C. The colonies were scraped with disposable scrapers and suspended in 2YT liquid medium supplemented with 100 μg/ml carbenicillin. The slurries were divided to aliquots and kept at −75° C. Two libraries were constructed, one from the spleen mRNA and one much smaller from hybridoma mRNA.

Biopanning

25 cm² flasks were coated with 20 μg/ml avidin (Sigma) in 0.1M NaHCO₃, pH 9.0, overnight at 4° C. A biotinylated peptide composed of amino acids 1-16 of the Aβ peptide at 1 μg/ml was applied to the coated flasks followed by blocking with 10% milk powder in PBS at room temperature.

Phages were prepared from each library by diluting 50 μl aliquots into 10 ml 2YT with 100 μg/ml carbenicillin and 0.2% glucose. Bacteria were grown to late-log phase and infected with 1×10¹⁰ cfu of helper phage M13KO7 (New England Biolabs, Beverly, Mass.), for 30 minutes at 37° C. The infected bacteria were centrifuged and the supernatant which contained non-infected phages was discarded. The cells were suspended in fresh 2YT with 100 μg/ml Carbenicillin, 50 μg/ml Kanamycin and 0.2 mM IPTG, and grown over night at 37° C. Phages were extracted as described elsewhere, precipitated with PEG-NaCl and suspended in 2 ml PBS and 5% blocker. Phages were applied to the previously blocked flasks and allowed to bind the peptide for 2 hrs at 37° C. Flasks were stringently washed with PBST (0.1%-1% TWEEN 20) and PBS. Late-log-phase growing TG1 cells were added and allowed to be infected with phages that bound the antigen. Aliquotes of the infected bacteria were diluted and plated on carbenicillin plates to estimate the titer of bound phage, and the rest of bacteria were used for further biopanning rounds.

Individual colonies that grew on selection plates were used to produce phages that were retested for antigen binding (by ELISA). Positive clones were kept for further analysis and as possible candidates for vaccine application and soluble protein production.

ELISA

Phages produced from individual bacteria were tested for their ability to bind either the biotinylated 1-16 peptide or beta amyloid peptide 1-40. Wells of microtiter plates (Maxisorb, Nunc) were coated with 20 μg/ml avidin (Sigma) in 0.1 M NaHCO₃ pH 9.0 over-night at 4° C.

The wells were blocked with 5% milk powder in PBS for 1 hr at 37° C. Phages displaying ScFv candidates were diluted to 10¹²/ml in 1% blocking solution and applied to the wells for 1 hr at 37° C. The wells were washed 3 times with PBST (0.05% TWEEN 20) and 3 times with PBS. Mouse anti-phage antibodies were applied at 1 μg/ml followed by a secondary anti-mouse horse-radish peroxidase (HRP)-conjugated antibody. HRP activity was detected at room temperature for 20 minutes using Ortophenylenediamine (OPD) reagent and measured at 492 nm with reference reading at 405 nm. Helper phage was used as a negative control and anti beta-amyloid-peptide antibody for positive control. For testing the binding of candidate phages to beta amyloid peptide, soluble AβP 1-40 (Sigma) was diluted to 10 μg/ml in sterile dH₂O and 50 μl aliquotes were distributed in microtiter plates and incubated over night at 37° C. The next day, the plates were washed, blocked and the detection of positive clones was carried out as described above.

Immunostaining

Phages displaying positive ScFvs were used to stain paraffin embedded brain sections of hAPP transgenic mice that contained amyloid plaques. Phages were diluted to 10¹²/ml in PBS and were overlaid on sections that were previously blocked with Histamouse (Zymed/Invitrogen, Carlsbad, Calif.). Mouse anti-phage antibodies were added at a concentration of 0.1 μg/ml and polymer (Zymed/Invitrogen) was used as a detecting tool. HRP reaction was develop using DAB. The sections were visualized for plaque staining using Leica DMLB microscope and Images were photographed with a ProGress C14 color video camera.

Soluble ScFv Constructs and Purification

Selected scFvs were removed by NcoI and NotI digestion and cloned in pMAL vector (New England Biolabs) that was modified to include an NcoI site in its multiple cloning site (MCS). This translational fusion caused the cloned scFvs to form a single protein with the maltose binding protein at their N termini. The plasmids were transformed into E. coli strain BL21-trxB. Single colonies were grown in 100 ml 2YT supplemented with 100 μg/ml carbenicillin and 1% glucose, to mid-log phase (OD 0.5 at 600 nm). IPTG at 1 mM final concentration was added and the cells kept growing for 3 hr at 32° C. The cells were harvested and suspended in column buffer (20 mM Tris-HCl, pH 7.4; 200 mM NaCl; 1 mM EDTA) with Complete Mini EDTA free (protease inhibitor) (Roche). They were sonicated and centrifuged. The supernatant was collected and filtered through 0.45 mm filter. The crude extract was loaded on manually packed amylose column and protein chromatography was carried out using Akta Prime (Amersham Biosciences). MBP-fused scFvs and MBP alone were eluted into fraction collector using elution buffer (column buffer plus 0.3 mM maltose).

Results Positive Phage-ScFv Selection

The spleen ScFv library contained an estimated number of 5×10⁵ clones, about 6% of which were self-ligated vector. Four rounds of biopanning were performed. The first round started with an approximate number of 3×10¹² phages and ended with a calculated number of 485,000 cfu (based on plating of diluted bacteria). At each additional panning cycle the phage titer was about 10 fold lower and 270 Colonies from each panning cycle were examined by ELISA and candidates that showed high absorbance at 492 nm (>0.1 OD), compared to helper phage control (0.003-0.005 OD), were analyzed by PCR using primers S1 and S6 of pCANTAB5E. Candidates which contained full length ScFv fragment were also digested with NheI to verify correct assembly of the heavy and light chains. A total of thirty final candidates were sequenced. The best clone was designated Q-RCK37 and used in immunostaining of AβP plaques. The hybridoma library contained a few thousand clones. One cycle of panning was performed and 30 colonies were digested and divided into 2 groups. One group consisted of 28 identical clones and the other group of 2 identical clones. The latter differ in a small insertion in the light chain. One out of the 28 identical clones was designated Q-RCK22. The nucleotide and amino acid sequences of RCK37 and RCK22 as well as their CDR sequences are shown in FIGS. 3A-3D. An ELISA analysis that compares its binding together with Q-RCK37 and helper phage as a control is shown in FIG. 4.

Immunohistology

Phages Q-RCK37, Q-RCK22 and helper phage as a control, were used to stain plaques of transgenic mice that express mutation V717F in APP and the Swedish mutation in the β-secretase cleavage site on APP. These mice develop a high number of plaques in their brains. Q-RCK22 stained mostly plaques. Q-RCK37 stained plaques and what is suspected to be neurofibrillary tangles. Most of the staining occurred in the cortex section of the brain (FIGS. 5A-5C).

Soluble ScFv

Previous studies demonstrated that the maltose binding protein stabilizes foreign proteins when expressed in E. coli and helps them to maintain being soluble in the bacterial cytoplasm (Bach et al., 2001). RCK37 and RCK22 were fused to the MBP gene and expressed in E. coli BL21-trxB strain which possesses a thioredoxin-reductase mutation. This strain of bacteria facilitates cytoplasmic disulfide-bond formation which increases the fraction of properly folded protein. Following expression of RCK37 and RCK22, RCK37 and RCK22 were purified by affinity chromatography on amylose resin and were tested by ELISA for binding to AβP. Both soluble ScFvs bound AβP were compared to MBP alone, which served as a control (FIG. 6).

Example 2 Single Domain Antibodies Against β-Amyloid Peptide

In order to minimize the size of scFv37 antibody, the heavy and light chains of scFv were cloned separately in a pET28 vector and each molecule was expressed in an E. coli BL21 lysS strain. The proteins were extracted from inclusion bodies and checked by ELISA for the binding of AβP (1-40) that was preaggregated overnight, and also for the binding of amino acids 1-16 of AβP.

Materials and Methods Cloning

The heavy and light chain of RCK37 were synthesized and amplified by PCR using the following primers in Table 2 below:

TABLE 2 HEAVY CHAIN 37H-fwd: 5′-CATATGGCTCATGTCCAG-3′ (SEQ ID NO: 40) 37h-REV: 5′-CTCGAGTGCAGAGACGGTGAC-3′ (SEQ ID NO: 41) LIGHT CHAIN 37L-fwd: 5′-CATATGGAGATAATGATAACGCAG-3′ (SEQ ID NO: 42) 37L-rev: 5′-CTCGAGGCGTTTCATCTCCAG-3′ (SEQ ID NO: 43)

PCR was carried out with Qiagen enzyme and a scFv37-carrying plasmid as a template DNA, using the following protocol: initial denaturation, 2 min at 94° C., followed by 25 cycles of 30 sec denaturation at 94° C., annealing for 45 sec at 48° C.; and 1 min DNA synthesis at 72° C. The final products contained the net nucleotide sequence of the heavy and light chains of RCK37. The PCR fragments were cloned in pGEM-T vector (Promega, Madison, Wis.), and the fragments were excised by NdeI and XhoI digestion and cloned in pET28 vector (Novagen, Madison, Wis.) in which the BglII-XhoI fragment was exchanged with the same fragment from pET21. This exchange enables the heavy or light chains to be fused to the His tag at the C-terminus only. The resulting ligated DNA molecules were transferred to E. coli strain BL21 lysS (Novagen). For heavy or light chain production, cells were grown in 1 liter culture at 37° C. to late-mid log phase (OD600=0.6). IPTG induction was started at this stage, using 0.8 mM IPTG overnight at 30° C. The next day, cells were centrifuged, and inclusion bodies were prepared as described in Biotechniques, Vol XI, No. 6, December 1991, pp 748-752. The purified heavy and light chains were dialyzed overnight in PBS to exchange the renaturation buffer in which they were suspended in the last step of inclusion body extraction. Protein concentrations were determined and the binding of the heavy and light chains to Aβp 1-40 was determined by ELISA.

ELISA

β-amyloid peptide 1-40, at increasing concentrations (0-5 μM) was applied to 96 well plate in duplicates of 50 μl each and incubated in 37° C. overnight. Wells were washed and blocked with 3% milk powder in PBS for 1 hr at 37° C. The 37H or 37L molecules were applied at the constant concentration of 12.5 μM, and 10D5 (monoclonal antibody against the N terminus of Aβ peptide) was also applied as a control at 1 μg/ml, for 1 hr at 37° C. Detection of 37H or 37L was carried out with mouse anti-H is antibody and with anti-mouse antibody for 10D5, followed by Goat-anti mouse antibody conjugated to HRP (horse radish peroxidase) for all three antibodies. In another assay, a constant concentration of 10 μM β-amyloid peptide 1-40 was applied to 96 well plate and the 37H or 37L molecules were applied at different concentrations (from 3.125 μM to 25 μM). The rest of the ELISA assay was performed as described above.

ThT

In vitro fibril formation of Aβp was measured by the Thioflavin T (ThT) binding assay. ThT binds amyloid fibrils and exhibits enhanced fluorescence emission at 485 nm upon excitation at 435 nm. Fluorescence intensity is correlated with the degree of Aβp fibril formation. Aβp was solubilized in dH₂O (pH 5.5) to 23.2 μM, aliquoted into 20 μl samples in blocked tubes and incubated in a 37° C. humidified incubator for a week. For disaggregation assay, 20 μl aliquote of 12.5 μM 37H, 25 μM 37H or 1 μM mAb 10D5 were added at this point to the Aβp aliquots, mixed well and incubated together for an additional week. To measure fluorescence as a measure of the amount of fibrils, 0.98 ml aliquots of 2 μM ThT (in 50 mM glycine, pH 9.0) were added to 20 μl Aβp preparations and read in an LSB-50 Perkin Elmer spectorofluorimeter.

Results

FIGS. 7A and 7B show the results of the ELISA assay performed with constant β-amyloid concentrations and elevated 37H or 37L concentrations. It demonstrates that both 37H and 37L bind Aβp 1-40 (Abeta 1-40), however the binding of 37H is 5 times stronger than the binding of 37L. The binding is linear at the concentrations examined, suggesting a real affinity of the antibody to β-amyloid peptide. The 10 mM β-amyloid peptide used was high enough to bind even the highest antibodies concentration used.

The same linear reactivity is observed when the concentrations of the antibodies are constant (12.5 μM), while the antigen's concentrations are titrated (FIGS. 7C and 7D); however, in this case, the binding is linear only up to 1.25 μM Aβp. In the two other concentrations examined, 2.5 and 5 μM, the binding was slightly lower (not shown), suggesting that the β-amyloid above 1.25 μM is aggregated and the binding is reduced as a result. This probably happened also in the assay presented in FIGS. 7A and 7B, but because 10 μM Aβ were used, there were enough linear β-amyloid molecules to bind the antibodies that were applied. The binding of antibody 10D5 is not shown in FIGS. 7A-7D.

In FIGS. 8A and 8B, the binding of 37H to β-amyloid fibrils in solution and their partial disaggregation is shown. Compared to the monoclonal antibody 10D5, the concentrations needed to solubilized the Aβ fibrils are relatively higher, but producing 37H is much easier and economical. Table 3 below provides a summary of the relevant aggregation values and the values of disaggregation.

TABLE 3 Percent Aggregation Percent Disaggregation A beta only 100 0 Plus 25 μM 37H 52.95 47.04459 Plus 12.5 μM 37H 62.29 30.71043 Plus 1 μM 10D5 61.35848357 38.64152

Example 3 In Vivo Experiments Material and Methods

TAPIR Assay with Nanobody 37H

Soluble nanobody 37H (single domain antibody) was used to stain brain sections of a PDAPP transgenic mice. Paraffin sections (5 μm) were deparaffinized by a series of xylenes, hydrated with a gradient series of ethanol, and quenched by 3% H₂O₂ in methanol and then blocked with Histamouse kit blocker A (Zymed, USA). 37H protein was diluted to 10 μg/ml PBS and overlaid on the sections for 2 hr at room temperature. Mouse anti-His tag-HRP conjugated antibodies were added at a concentration of 0.1 mg/ml. Slides were developed with DAB (Zymed).

Animals

Male and female PDAPP mice and non-Tg littermates were used. The mice were genetically engineered by employing a platelet-derived growth factor promoter driving a hβAPP minigene encoding the 717_(V→F) mutation associated with familial AD. The ambient temperature was maintained at 25° C.±1 and a 12:12-h light-dark cycle was maintained throughout the experiment. Food and water were available ad libitum. Animal care, maintenance and experimental procedures were according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Immunization with Phage-Anti EFRH scFv

The administration of scFv-phage to the mice was started when they were nine months old. Each mouse of the scFv-phage group received 10¹¹ phages per administration every 2 weeks for 2 months and then once a month for a total of 14 applications (administrations). Intranasal administration was done as follows: briefly, the mouse was held firmly with its head pointing to the ceiling, with one hand, and with the other hand, phages were applied using a 100-μl pipette with narrow sterile protected tips that contained 20 μl of solution. Approximately 10 μl per nostril were applied, in short intervals to ensure that the solution wetted the mucosa of the nose. The scFv 22 and scFv 37 treated groups included 12 mice each. The PBS control group included 11 mice.

Phage 37 was also applied by intraperitoneally (IP) method, 10¹¹ phages in 300 μl were injected into each mouse peritoneum. The number of administrations was similar to the intranasal (IN) treatment. Five mice were included in this group.

Preparation of Brain Tissues

At the end of the experiment, the mice (20 months old) were euthanized, using an overdose of a standard inhalation of anesthesia (Isofluran®, Baxter, USA). Next, their brains were removed and the right hemispheres collected from each mouse were fixed for 24 hr in 4% paraformaldehyde/PBS, transferred to PBS (pH 7.4), and then immersed in 30% sucrose in PBS. When the brains sank, they were frozen in Acetone-dry-ice bath. Serial coronal sections (5 μm), in an anterior-to-posterior direction, 250 μm apart from each other, were prepared in a cryostat for histology. The brain pathology was examined after H&E staining and hemosiderin stainings were performed to detect any vascular hemorrhage.

Quantitative Analysis of Amyloid Plaque Load

Two well-defined coronal sections at the levels of −1.6 and −3.6 from bregma were selected for thioflavin-S staining (which stains dense plaques). Sections were hydrated, and stained first with hematoxylin to quench autofluorescence, and then with 1% thioflavin-S for 3 minutes, followed by immersion in 1% acetic acid for 20 minutes, then washed, cleared, and mounted. Images from these sections were captured by a CCD color video camera (ProgRes C14, Jenoptic, Jena, Germany) attached to a Leica DMLB microscope (Leica, Germany) and analyzed with appropriate software (Leica Qwin, Leica, Germany). The total amyloid dense core load in plaques in the cortex and hippocampus was expressed as the percentage of the area stained with thioflavin-S out of the total area of the these areas in each section.

Staining with 21F12, an Anti-Aβ 1-42 Antibody

This antibody stains soluble and fibrillar Aβ 1-42. Slides with serial brain sections were washed in TBS and quenched in 3% H₂O₂ in methanol for 15 min in room temp, followed by 3 washes in TBS. Denaturation was carried out in 90% formic acid for 30 min. at room temp, followed by 3 washes in TBS. Tissue permeabilization was done by 0.3% Triton in TBS for 20 min at room temp followed by 3 washes in TBS. Blocking was done with UV block (LabVision) for 10 min at room temp and antibody 21F12 was applied at 1:1000 in PBS for 1 hr at 37° C. followed by overnight incubation at 4° C. and 3 washes, 5 min each, in TBS. Supes Picture (Zymed, ready to use) was applied for 20 min at room temp with gentle shaking followed by 4 TBS washes, each for 4 min. Developing was done with DAB (Zymed) for 5 min and stopped with multiple dH₂O washes.

Results

FIG. 9 shows amyloid plaques of a PDAPP Tg mouse stained with 37H nanobody (the heavy chain of scFv RCK37). Note that the antibody stains plaques, fibrillar and soluble Aβ deposits in the tissue. In heavily condensed plaques, the nanobody stains the outskirts of the plaque that are less condensed. Around some plaques, glial cells that also were stained are visible, probably because they contain beta amyloid deposits.

In FIG. 10, a summary of the plaque load detected by thioflavin S staining in the cortex and hippocampus of PDAPP mice treated with phage 22 or phage 37, versus control mice, treated with PBS only, is presented. Phage 37 reduced plaque load significantly in both the cortex and hippocampus areas of mice treated with it by intranasal application. Intraperitoneal (IP) application of phage 37 did not show significant plaque load reduction. Plaque load reduction in phage 22 treated mice was much more dramatic both in the cortex and in the hippocampus, compared to untreated controls.

FIG. 3 shows a summary of the plaque load detected by staining with antibody 21F12 in the cortex and hippocampus of PDAPP mice treated with phage 22 or phage 37, versus control mice, treated with PBS only. Here too, both phage 37 and phage 22 reduced plaque load significantly in both the cortex and hippocampus areas of mice treated with these scFvs by intranasal application. IP application of phage 37 did not show significant plaque load reduction.

Both scFv 22 and scFv 37 significantly reduced the Aβ load in treated mice brains, They affected the soluble and fibrillary amyloid deposits (stained with 21F12 antibody) as well as the dense amyloid plaques (stained by thioflavin S). Furthermore, intranasal administration/application of the phage scFVs is an effective way to introduce the scFv antibodies to the mice brains, compared with the IP application of phage 37. While IP administration of phage did show reduced plaque load, the reduced plaque load was not as significant as by intranasal administration.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

All references cited herein, including journal articles or abstracts, published or corresponding U.S. or foreign patent applications, issued U.S. or foreign patents, or any other references, are entirely incorporated by reference herein, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited within the references cited herein are also entirely incorporated by reference.

Reference to known method steps, conventional methods steps, known methods or conventional methods is not in any way an admission that any aspect, description or embodiment of the present invention is disclosed, taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

Thus the expressions “means to . . . ” and “means for . . . ”, or any method step language, as may be found in the specification above and/or in the claims below, followed by a functional statement, are intended to define and cover whatever structural, physical, chemical or electrical element or structure, or whatever method step, which may now or in the future exist which carries out the recited function, whether or not precisely equivalent to the embodiment or embodiments disclosed in the specification above, i.e., other means or steps for carrying out the same functions can be used; and it is intended that such expressions be given their broadest interpretation.

REFERENCES

-   Bach H, Mazor Y, Shaky S, Shoham-Lev A, Berdichevsky Y, Gutnick D L,     Benhar I. Escherichia coli maltose-binding protein as a molecular     chaperone for recombinant intracellular cytoplasmic single-chain     antibodies. J Mol. Biol. (2001) 312(1):79-93 -   Bacskai B J, Kajdasz S T, McLellan M E, Games D, Seubert P, Schenk     D, Hyman B T. 3. Non-Fc-mediated mechanisms are involved in     clearance of amyloid-beta in vivo by immunotherapy. J. Neurosci.     2002 Sep. 15; 22(18):7873 -   Frenkel, D., Solomon B. and Benhar I. Modulation of Alzheimer's beta     amylois neurotoxicity by site-directed single-chain antibody. J. of     Neuroimmunology (2000) 106:23-31. -   Schenk D., Barbour R. Dunn W. et al. Immunization with amyloid-beta     attenuates Alzheimer-disease-like pathology in the PDAPP mouse.     Nature (1999) 400:173-177. 

1. An antibody against β-amyloid peptide, comprising the complementarity-determining regions (CDRs) of an antibody heavy chain or light chain selected from the group of CDR1, CDR2 and CDR3 sequences consisting of: (1) SEQ ID Nos: 22, 24 and 26; and (2) SEQ ID Nos: 28, 30 and
 32. 2. The antibody of claim 1, which is a single domain antibody.
 3. The antibody of claim 2, wherein the single domain comprises the CDR sequences of SEQ ID Nos: 22, 24 and
 26. 4. The antibody of claim 2, wherein the single domain comprises the CDR sequences of SEQ ID Nos: 28, 30 and
 32. 5. The antibody of claim 1, which is a single chain (scFv) antibody.
 6. The antibody of claim 5, comprising the CDR sequences of SEQ ID Nos: 22, 24, 26, 28, 30 and
 32. 7. The antibody of claim 6, comprising the amino acid sequence of SEQ ID NO:8.
 8. A nucleic acid molecule encoding the antibody of claim
 1. 9. The nucleic acid molecule of claim 8, comprising the nucleotide sequence of SEQ ID NO:7.
 10. A vector comprising the nucleic acid molecule of claim
 8. 11. The vector of claim 10, which is an expression vector capable of expressing in a host cell the antibody against β-amyloid peptide.
 12. A host cell transformed with the vector of claim
 11. 13. A method for producing and isolating an antibody against β-amyloid peptide, comprising: culturing the transformed host cell of claim 12 to express and produce an antibody against β-amyloid peptide; and isolating the produced antibody from the cell culture.
 14. A host cell transformed with the nucleic acid molecule of claim
 8. 15. A pharmaceutical composition comprising an effective amount of the antibody of claim 1 and a pharmaceutically acceptable carrier, diluent, excipient or auxillary agent.
 16. A method for reducing β-amyloid plaque load in the brain, comprising administering the antibody of claim 1 to a patient in need thereof.
 17. The method of claim 16, wherein the antibody is administered intranasally. 